The aim of this study was to establish the radiosensitising properties of gemcitabine in a pair of related bladder tumour cell lines with differential radiosensitivity. The radioresistant bladder tumour cell line MGH-U1 and its radiosensitive mutant clone, S40b (both p53 mutant), had SF(2) values (surviving fraction at 2 Gy) of 0.98 and 0.64, respectively (P<0.001). Colony-forming assays showed that at 0.01 microM gemcitabine radiosensitisation occurred only in the S40b cell line (dose-modifying factor (DMF)=1.4). At 0.3 microM (killing 50% of cells), both cell lines were radiosensitised; DMF=2.25 and 1.2 for MGH-U1 and S40b, respectively. These data suggest that gemcitabine is an effective radiosensitiser in bladder cancer cell lines, with greater sensitisation in the radioresistant parental line-a feature that should be useful in a clinical setting.

The use of definitive radiotherapy for treatment of invasive bladder cancer has the advantage of preserving bladder function, but tumour regression is only achieved in approximately 40-50% of patients. Knowledge of the molecular basis of sensitivity to ionizing radiation and identification of potential molecular predictors will provide useful information regarding patient response and thus help clinicians to individualize treatment. The recent application of cDNA expression array technology provides a useful tool to investigate hundreds or even thousands of genes in a single experiment. In our study, we have used the Atlas human stress cDNA array trade mark to investigate the expression profile of stress-related and DNA repair genes in a radioresistant bladder carcinoma cell line (MGH-U1) and its radiosensitive subclone (S40b). This provides an ideal situation to study genes related to radiation because the genotypes of both cell lines are basically similar and differential changes detected are likely to be related to the different radiosensitivity phenotype. Of 234 genes blotted on the array, 3 genes (Heat shock protein 90, Heat shock protein 27 and Nicotinamide N-methyl transferase) showed consistent downregulation in the radiosensitive clone in 2 independent experiments. These results were further confirmed for HSP27 and NNMT using Sybr Green I-based real-time QRT-PCR. The role of heat shock proteins (HSPs) in response to radiation remains to be determined; however, the results of our present work suggest a possible role of HSP27 in determining radiosensitivity. Our study also opens avenues for the investigation of genes, such as NNMT, which has not previously been linked to response to radiation.

The epidermal growth factor receptor (EGFR) is expressed in a wide variety of epithelial tumours including carcinoma of the bladder. Stimulation of the EGFR pathway is blocked by ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor. Radical radiotherapy is an established organ sparing treatment option for muscle invasive bladder cancer and this study has explored the possibility for the use of ZD1839 as a radiosensitiser in this scenario. The effect of combination treatment with ZD1839 (0.01 microM) and ionising radiation in the established bladder cancer cell lines MGH-U1 and its radiosensitive mutant clone S40b was measured by clonogenic assays. A highly significant radiosensitising effect was seen in both cell lines (P < 0.001 for MGH-U1 and S40b cell lines). This effect was independent of the concentration of the drug and the duration of exposure prior to treatment with ionising radiation. Cell cycle kinetics of both cell lines was not significantly altered with ZD1839 (0.01 microM) as a single agent. A modest induction of apoptosis was observed with ZD1839 (0.01 microM) as a single agent, but a marked induction was observed with the combination treatment of ZD1839 and ionising radiation. These results suggest a potentially important role for ZD1839 in combination with radiotherapy in the treatment of muscle invasive bladder cancer.

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.

Previous studies suggest that bladder cancer risk may vary with GST genotype but these results are inconsistent. The aim of this study was to explore whether GSTM1, GSTT1 and GSTP polymorphisms were associated with increased bladder cancer risk in an Egyptian population. GSTM1, GSTT1 and GSTP1 genotype frequencies were determined in bladder cancer cases (n=72) and healthy controls with no history of malignancies (n=82) using PCR-based techniques. The GSTT1*2 genotype was particularly associated with increased risk (OR 2.71, 95%CI 1.27-5.73) and the GSTM1*2 genotype to a lesser extent (OR 1.63, 95%CI 0.85-3.10). 18.1% of cases but only 7.3% of controls were GSTP1*B*B homozygotes (OR 2.38, 95%CI 0.83-6.87). The presence of two or more a priori at-risk genotypes was associated with increased bladder cancer risk (OR 2.42; 95%CI 1.47-3.97). These results suggest that polymorphisms in the GST genes are associated with increased risk of bladder cancer among Egyptians.

Mutation of human homologues of DNA mismatch repair (MMR) genes in tumours has been shown to be associated with the phenomenon of microsatellite instability (MSI). Several studies have reported the occurrence of MSI in bladder cancer, but evidence of involvement of MMR genes in the pathogenesis of this cancer is still unclear. We therefore utilized quantitative immunohistochemical (IHC) image analysis and PCR-based allelotype analysis to determine hMLH1 and hMSH2 genes alteration in a cohort of Egyptian bladder cancer samples. IHC analysis of 24 TCC and 12 SCC revealed marked- intra and intertumour heterogeneity in the levels of expression of the two MMR proteins. One TCC lost MLH1 expression and one lost MSH2, (1/24, 4%), and one SCC lost MSH2 (1/12, 8%). A large proportion of analysed tumours revealed a percentage positivity of less than 50% for MLH1 and MSH2 expression (44% and 69%, respectively). Complete loss of heterozygosity in three dinucleotide repeats lying within, or in close proximity to, hMLH1 and hMSH2 was rare (2/57, (4%) for MLH1; and 1/55, (2%) for MSH2), however allelic imbalance was detected in 11/57 (hMLH1) and 10/55 (hMSH2) at any of the informative microsatellite loci. These alterations in structure and expression of DNA MMR genes suggest their possible involvement in the tumorigenesis and/or progression of bladder cancer.

Schistosoma haematobium-infected patients are more likely to develop bladder cancer and be more exposed to carcinogenic N-nitroso compounds than uninfected patients. As N7-methylguanine is a marker of exposure to methylating agents of this type, we have measured N7-methyldeoxyguanosine 3'-monophosphate (N7-MedGp) by (32)P postlabeling. DNA was isolated from 42 paired normal and tumor tissue of Egyptians with bladder cancer. N7-MedGp was detected in DNA from 93% of the tumors and 74% of the normal bladder tissue samples. Adduct levels were highly variable and ranged from 0.04 to 6.4 and from 0.02 to 0.72 micromol/mol deoxyguanosine 3'-monophosphate (dGp) in tumor and normal DNA, respectively. N7-MedGp levels in normal and tumor DNA were highly correlated with one another (P = 0.007). The mean difference (95% confidence interval) in adduct levels between tumor and normal DNA was 0.21 (0.13-0.32) micromol/mol dGp and this was statistically significant (P < 0.001). The adduct ratio (tumor DNA/normal DNA) varied between 0.2 and 136 (median, 4.6). N7-MedGp levels were not associated with gender, age, or the presence of schistosomiasis. However, lower N7-MedGp levels were found in normal DNA from individuals lacking the GSTM1 gene (P = 0.03) but not the GSTT1 gene or in subjects with the Ile105Val GSTP1 polymorphism. These results show that exposure to methylating agents is widespread and suggest that such exposure may play a role both in tumor initiation and progression.

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